Bottom Line:
Furthermore, both LPS and domoic acid did not significantly affect TGF-beta1 gene expression and protein release.However, in contrast, no statistically significant increase in MMP-9 expression and release was observed after domoic acid treatment.Taken together our observations do not support the hypothesis that a short term (4 to 24 hours) in vitro exposure to domoic acid, at a concentration toxic to neuronal cells, activates rat neonatal microglia and the concomitant release of the pro-inflammatory mediators tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinases-9 (MMP-9), as well as the anti-inflammatory cytokine transforming growth factor beta1 (TGF-beta1).

ABSTRACTThe excitatory amino acid domoic acid is the causative agent of amnesic shellfish poisoning in humans. The in vitro effects of domoic acid on rat neonatal brain microglia were compared with E. coli lipopolysaccharide (LPS), a known activator of microglia mediator release over a 4 to 24 hour observation period. LPS [3 ng/mL] but not domoic acid [1 mM] stimulated a statistically significant increase in TNF-alpha mRNA and protein generation. Furthermore, both LPS and domoic acid did not significantly affect TGF-beta1 gene expression and protein release. Finally, an in vitro exposure of microglia to LPS resulted in statistically significant MMP-9 expression and release, thus extending and confirming our previous observations. However, in contrast, no statistically significant increase in MMP-9 expression and release was observed after domoic acid treatment. Taken together our observations do not support the hypothesis that a short term (4 to 24 hours) in vitro exposure to domoic acid, at a concentration toxic to neuronal cells, activates rat neonatal microglia and the concomitant release of the pro-inflammatory mediators tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinases-9 (MMP-9), as well as the anti-inflammatory cytokine transforming growth factor beta1 (TGF-beta1).

f2-md503113: RT-PCR analysis of TNF-α gene expression in rat neonatal microglia after in vitro treatment with LPS or DOM. Neonatal rat brain microglia (2.8–5.0 x 106 cells/culture dish) were treated with (A) LPS [3 ng/mL] or (B) DOM [1mM] for 4 to 24 hours as described in Experimental. Amplification of TNF-α and S12 mRNAs shows the predicted cDNA size after separation on a 1.5 % agarose gel and visualization by SYBRR Gold nucleic acid staining. The S12 ribosomal RNA gene product was amplified for 25 and 30 cycles and demonstrated equal loading of the gels. Quantification of the TNF-α product was done after 25 cycles, where mRNA expression was observed to be in the linear range. The figure depicts one of two similar experiments.

Mentions:
We have previously reported that in vitro stimulation of microglia with LPS [10ng/mL] will result in TNF-α mRNA expression [27]. As shown in Figure 2A, while untreated (control) microglia did not express TNF-α, stimulation with LPS [3 ng/ml] resulted in a time-dependent increase in TNF-α mRNA expression relative to controls as determined by semi-quantitative RT-PCR within the linear range of amplification (25 cycles) after 4 hours. In contrast, as shown in Figure 2B, domoic acid [1mM] induced only minor changes in TNF-α mRNA expression at the 4 hour time point.

f2-md503113: RT-PCR analysis of TNF-α gene expression in rat neonatal microglia after in vitro treatment with LPS or DOM. Neonatal rat brain microglia (2.8–5.0 x 106 cells/culture dish) were treated with (A) LPS [3 ng/mL] or (B) DOM [1mM] for 4 to 24 hours as described in Experimental. Amplification of TNF-α and S12 mRNAs shows the predicted cDNA size after separation on a 1.5 % agarose gel and visualization by SYBRR Gold nucleic acid staining. The S12 ribosomal RNA gene product was amplified for 25 and 30 cycles and demonstrated equal loading of the gels. Quantification of the TNF-α product was done after 25 cycles, where mRNA expression was observed to be in the linear range. The figure depicts one of two similar experiments.

Mentions:
We have previously reported that in vitro stimulation of microglia with LPS [10ng/mL] will result in TNF-α mRNA expression [27]. As shown in Figure 2A, while untreated (control) microglia did not express TNF-α, stimulation with LPS [3 ng/ml] resulted in a time-dependent increase in TNF-α mRNA expression relative to controls as determined by semi-quantitative RT-PCR within the linear range of amplification (25 cycles) after 4 hours. In contrast, as shown in Figure 2B, domoic acid [1mM] induced only minor changes in TNF-α mRNA expression at the 4 hour time point.

Bottom Line:
Furthermore, both LPS and domoic acid did not significantly affect TGF-beta1 gene expression and protein release.However, in contrast, no statistically significant increase in MMP-9 expression and release was observed after domoic acid treatment.Taken together our observations do not support the hypothesis that a short term (4 to 24 hours) in vitro exposure to domoic acid, at a concentration toxic to neuronal cells, activates rat neonatal microglia and the concomitant release of the pro-inflammatory mediators tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinases-9 (MMP-9), as well as the anti-inflammatory cytokine transforming growth factor beta1 (TGF-beta1).

ABSTRACTThe excitatory amino acid domoic acid is the causative agent of amnesic shellfish poisoning in humans. The in vitro effects of domoic acid on rat neonatal brain microglia were compared with E. coli lipopolysaccharide (LPS), a known activator of microglia mediator release over a 4 to 24 hour observation period. LPS [3 ng/mL] but not domoic acid [1 mM] stimulated a statistically significant increase in TNF-alpha mRNA and protein generation. Furthermore, both LPS and domoic acid did not significantly affect TGF-beta1 gene expression and protein release. Finally, an in vitro exposure of microglia to LPS resulted in statistically significant MMP-9 expression and release, thus extending and confirming our previous observations. However, in contrast, no statistically significant increase in MMP-9 expression and release was observed after domoic acid treatment. Taken together our observations do not support the hypothesis that a short term (4 to 24 hours) in vitro exposure to domoic acid, at a concentration toxic to neuronal cells, activates rat neonatal microglia and the concomitant release of the pro-inflammatory mediators tumor necrosis factor-alpha (TNF-alpha) and matrix metalloproteinases-9 (MMP-9), as well as the anti-inflammatory cytokine transforming growth factor beta1 (TGF-beta1).